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Circadian Dysregulation Is Associated with Alterations in Tumor Suppressor Activity in Murine Mammary Tissue

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Circadian Dysregulation Is Associated with Alterations in Tumor Suppressor Activity in Murine Mammary Tissue Circadian Dysregulation is Associated with Alterations in Tumor Suppressor Activity in Murine Mammary Tissue by Hiruni M. Aponso A THESIS submitted to Oregon State University Honors College in partial fulfillment of the requirements for the degree of Honors Baccalaureate of Science in Biochemistry and Molecular Biology (Honors Scholar) Presented May 29, 2020 Commencement June 2020 AN ABSTRACT OF THE THESIS OF Hiruni M. Aponso for the degree of Honors Baccalaureate of Science in Biochemistry and Molecular Biology presented on May 29, 2020. Title: Circadian Dysregulation is Associated with Alterations in Tumor Suppressor Activity in Murine Mammary Tissue. Abstract approved:_____________________________________________________ Patrick Chappell Breast cancer is the second most common cancer among women in the US, however, only a small fraction of cases are attributable to heritable genetic mutations; the bulk arises from various behavioral and environmental factors. Loss of functional p53 is observed in over 50% of cases, and its activity relies on post-translational acetylation or deacetylation by various cellular proteins. SIRT1 is a class III HDAC that can remove the activating acetyl moiety. In a normal cell, stress activates p53, which then transcribes Hic1. HIC1 represses transcription of Sirt1 to prevent it from deactivating P53 by removing its acetyl group. Hic1 is epigenetically regulated, and hypermethylation of the Hic1 promoter can result in silencing of its expression. Previous work in our lab demonstrated increased methylation in the promoter region of Hic1 in mammary tissue derived from mice exposed to extended light at night (LAN) Due to an established link between SIRT1 and the regulation of the circadian rhythm, the present study explores the abundance of P53, SIRT1, and phosphorylated SIRT1 (p-SIRT1) in response to circadian dysregulation. Our results indicate a unique abundance pattern of SIRT1 and P53 in normal murine mammary tissue as well as alterations in P53, SIRT1, and p-SIRT1 abundance after a three-week period of exposure to LAN. Key Words: Circadian clock, SIRT1, P53, breast cancer, epigenetics Corresponding e-mail address: [email protected] ©Copyright by Hiruni M. Aponso May 29, 2020 Circadian Dysregulation is Associated with Alterations in Tumor Suppressor Activity in Murine Mammary Tissue by Hiruni M. Aponso A THESIS submitted to Oregon State University Honors College in partial fulfillment of the requirements for the degree of Honors Baccalaureate of Science in Biochemistry and Molecular Biology (Honors Scholar) Presented May 2020, 2020 Commencement June 2020 Honors Baccalaureate of Science in Biochemistry and Molecular Biology project of Hiruni M. Aponso presented on May 29, 2020. APPROVED: _____________________________________________________________________ Patrick Chappell, Mentor, representing Biomedical Sciences _____________________________________________________________________ Maude David, Committee Member, representing Microbiology _____________________________________________________________________ Kate Shay, Committee Member, representing Biochemistry and Biophysics _____________________________________________________________________ Toni Doolen, Dean, Oregon State University Honors College I understand that my project will become part of the permanent collection of Oregon State University, Honors College. My signature below authorizes release of my project to any reader upon request. _____________________________________________________________________ Hiruni M. Aponso, Author Introduction Many industries and services rely on around-the-clock operations that require night-shift work.1,2 Shift work is associated with a number of health problems including sleep disturbances, cardiovascular dysfunction, metabolic syndrome, oxidative stress, and breast cancer.1-4 Under normal conditions, body functions of the respiratory, digestive, and cardiovascular systems rhythmically fluctuate throughout the day in time with the sleep/wake cycle.2,5 Many essential biological processes occur during sleep that are critical for overall health. When the normal sleep/wake cycle is inverted, our bodies are forced to perform activities during the dark phase, causing a shift in the body’s daily rhythmicity.2,5 The continuous stress of long work hours and nighttime light exposure eventually misaligns the internal clock with the sleep/wake cycle, leading to negative biological effects.1,2,5 The circadian clock is a light-entrainable timing system present in most cells that regulates and coordinates physiological processes by temporally inducing expression of target genes.6-9 In mammals, the master circadian clock is found in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus.7,9 The clock oscillates with a 24 h periodicity which can be modulated by intercellular signals and light input.6-10 Functionally, oscillation relies on positive and negative feedback loops generated from the transcription factors Brain and Muscle ARNT-like protein-1 (BMAL1) and Circadian Locomotor Output Cycles Kaput (CLOCK).6-10 CLOCK and BMAL1 form an active heterodimer which transcribes multiple genes, including the core clock genes Cryptochrome (Cry1 and Cry2) and Period (Per1, Per2, and Per3).6-10 Once CRY and PER proteins have accumulated, they form a heterodimer that interacts with CLOCK:-BMAL1 to suppress its activity, in turn repressing their own transcription (Figure 1).6-10 This positive- negative feedback loop has a cyclic nature in which the CLOCK:-BMAL1 complex is the most active during the day, and CRY:-PER inhibition peaks during the night.6-10 CLOCK also possesses histone acetyltransferase (HAT) activity, which facilitates chromatin remodeling events.11,12 In addition, CLOCK is able to acetylate non-histone proteins, including its own binding partner BMAL1 (Figure 1).11,12 In parallel with the suppression of clock- controlled gene transcription, CLOCK acetylates BMAL1 at a conserved Lys537 residue. This acetylation directs CRY to the CLOCK:-BMAL1 complex and instigates the repression of transcriptional activity (Figure 1).11,12,13 To counterbalance BMAL1 acetylation, histone deacetylase (HDAC) SIRT1 removes the acetyl group from Lys537 allowing the CLOCK:- BMAL1 complex to resume transcriptional activity (Figure 1).11,12 Figure 1. The regulatory mechanism of the circadian clock. SIRT1 deacetylates BMAL1 at Lys537 allowing the CLOCK:-BMAL1 heterodimer to bind to E-box elements in the promoter regions of clock-controlled genes (CCGs) including cry and per. CRY and PER proteins accumulate and form a dimer. The HAT activity of CLOCK acetylates BMAL1 at Lys537 and thereby directs the CRY:-PER dimer to the CLOCK:-BMAL1 complex. The CRY:-PER dimer inhibits CLOCK-BMAL1 activity and restarts the transcriptional cycle. SIRT1 is the mammalian homolog of yeast Sir2 and is a NAD+-dependent deacetylase involved in metabolic and physiological processes.7,10-12,14 The SIRT1 enzymatic reaction involves hydrolysis of NAD+ and transfer of the target protein’s acetyl group to the 2’-OH position of ADP-ribose, producing nicotinamide and O-acetyl-ADP-ribose.7,14 Since SIRT1 exclusively utilizes NAD+ as a co-substrate, its activity is dependent on the availability of NAD+.7,11,12,14 The rate-limiting step of the NAD+ salvage pathway is performed by nicotinamide phosphoribosyltransferase (NAMPT), and SIRT1 activity correlates with NAMPT abundance.11,12,14 CLOCK:-BMAL1 regulate induction of Nampt transcription, resulting in oscillatory abundance of NAMPT.11,12 Thus, because the NAD+ salvage pathway is circadian- regulated by the clock, the activity of SIRT1 is modulated in a circadian manner in phase with CLOCK:-BMAL1 activity.7,11,12 Figure 2. Effects of epigenetic silencing of HIC1 on the DNA-damage response. Silencing of HIC1 allows the transcription of SIRT1. SIRT1 HDAC activity can inhibit P53 activity. Baylin, S. B., & Ohm, J. E., 19 Nature Reviews Cancer, 2006. SIRT1 is a class III HDAC with the ability to deacetylate both histone and non-histone proteins, including the key tumor suppressor P53.12,15 Under normal cell conditions, P53 remained inactive at low concentrations.16 In response to DNA damage, P53 is rabidly induced and post-translationally modified to the active acetylated form which can direct transcription of proapoptotic genes. (Figure 2)6,12 Hic1 (Hypermethylated in Cancer 1), a P53 target gene, is transcriptional repressor which inhibits the transcription of SIRT1, preventing SIRT1-mediated deacetylation of Ac-P53 (Figure 2).15 Mutations or epigenetic silencing that disrupts P53 activity is often found in cancer patients, suppressing the ability to undergo cell cycle arrest, which then results in malignant cell growth. Preliminary studies conducted at the Chappell lab has indicated changes in transcript levels of Hic1 and Sirt1 as well as increases in methylation at the Hic1 loci after prolonged exposure to light-at-night (LAN) (Figure 3). Exposure to 3wks of LAN caused an increase in promoter methylation of the Hic1 locus (Figure 3a), and differential expression of Hic1 transcripts assessed by qPCR (Figure 3b). These observations were accompanied by insignificantly changed transcription of Sirt1 at all Zeitgeber times (ZT), with ZT times being time of day cues, after 3wks LAN (Figure 3c), but significant upregulation of Sirt1 transcription in mice exposed for 6wks LAN (Figure 3d). We believe extended exposure to LAN will cause circadian dysregulation and thereby induce epigenetic silencing of
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